Sphingolipids are important mediators and regulators of cell signaling pathways. Our studies have focused on the actions of two classes of sphingolipids represented by glycosphingolipids (GSLs) and sphingosine-1-phosphate. Our work is aimed at defining the normal functions of these sphingolipids and understanding their roles in disease processes. GSLs are found in the outer leaflet of the plasma membrane and are concentrated in specialized signaling structures. They are particularly abundant in neuronal cells in the form of gangliosides (sialic acid containing GSLs). Through genetic disruption of genes that encode synthetic enzymes for GSLs, we have created a series of mice that express limited glycosphingolipid structures. We are using these mice to discover the functions of GSLs. When the cellular machinery responsible for GSL degradation is defective, GSL storage diseases result in which profound neurodegeneration occurs. Examples are Tay-Sachs and Gaucher diseases. We are attempting to understand how the accumulation of GSLs cause neurodegeneration through the construction of animal models of the diseases. Our major accomplishments this year include the establishment of mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide alpha2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. We also continued our studies on the G-protein coupled receptor for sphingosine-1-phosphate, S1P1. We had previously shown the the global knockout of S1P1 in mice caused defective coverage of blood vessels by vascular smooth muscle cells (VSMCs). Since S1P1 receptor expression is not restricted to a particular cell type, it was not known whether the S1P1 receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in endothelial cells (ECs). By using the Cre/loxP system, we disrupted the S1P1 gene solely in ECs. The phenotype of the conditional mutant embryos mimicked the one obtained in the embryos globally deficient in S1P1. Thus, vessel coverage by VSMCs is directed by the activity of the S1P1 receptor in ECs.